A simple co-culture system for generation of embryonic stem like cells from testis

الملخص EN

Background : New research proposes the pluripotency of spermatogonial cells obtained from testis.

These spermatogonia-derived stem cells are called embryonic stem like cell that express embryonic stem cell markers and differentiate to the three germ layers. Objectives : The aim of this study was the generation of embryonic stem like cells from neonatal mouse testis. Materials and Methods : Testis cells were collected from neonatal mouse.

After decapsulation, testis was mechanically dissected and dissociated via a two-step mechanical and enzymatic digestion.

The spermatogonia and sertoli cells were cultured together in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 15 % FBS and LIF.

Before one week, several small spermatogonia colonies were observed on top of the monolayer of sertoli cells.

These colonies were passaged every four days.

ES Like cells colonies that resembled ES cell was appeared within 2-3 weeks (at passages 5).

Real time PCR was performed to analyze the expression of a subset of pluripotency markers, as well as germ cell-specific genes.

ES Like cells were confirmed with SSEA1, SOX2 and Oct4 immunofluorescence stainng as pluripotency markers. Results : Results showed that at fifth passages, the pluripotency genes; Nanog and c-myc has significant increase in ES Like cells in compare with spermatogonia cells, whereas the spermatogonial markers ; Stra8, mvh, and piwill2 became downregulated.

In addition to these pluripotency genes, the ES cell marker SSEA-1, SOX2 and Oct4 was expressed in the ES-like cells, similar to ES cells. Conclusions : These researh indicates pluripotency evidence of ES-like cells derived from testis.

ES-like cells shows some molecular characteristics with embryonic stem cells.